Canine interleukin-4 receptor alpha antibodies

ABSTRACT

The present invention provides antibodies to canine IL-4 receptor alpha that have a high binding affinity for canine IL-4 receptor alpha, and that can block the binding of canine IL-4 and/or IL-13 to canine IL-4 receptor alpha. The present invention further relates to epitopes of IL-4 receptor alpha that bind to the antibodies to canine IL-4 receptor alpha. The present invention further provides the use of the antibodies for the treatment of atopic dermatitis in dogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) of provisional applications U.S. Ser. No. 63/015,220 filed Apr. 24, 2020, U.S. Ser. No. 63/015,209 filed Apr. 24, 2020, U.S. Ser. No. 62/951,778, filed Dec. 20, 2019, and U.S. Ser. No. 62/951,793, filed Dec. 20, 2019, the contents of all of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies to canine IL-4 receptor alpha that have a high binding affinity for canine IL-4 receptor alpha, and that can block the binding of canine IL-4 and/or IL-13 to canine IL-4 receptor alpha. The present invention further relates to epitopes that bind to the antibodies to canine IL-4 receptor alpha. The present invention also relates to use of the antibodies of the present invention in the treatment of atopic dermatitis in dogs.

BACKGROUND OF THE INVENTION

The immune system comprises a network of resident and recirculating specialized cells that function collaboratively to protect the host against infectious diseases and cancer. The ability of the immune system to perform this function depends to a large extent on the biological activities of a group of proteins secreted by leukocytes and collectively referred to as interleukins. Among the well-studied interleukins are four important molecules identified as interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31), and interleukin-22 (IL-22). IL-4 and IL-13 are closely related proteins that can be secreted by many cell types including CD4⁺ Th2 cells, natural killer T cells (NKT), macrophages, mast cells, and basophils. IL-4 and IL-13 display many overlapping functions and are critical to the development of T cell-dependent humoral immune responses. It is known that IL-4 binds with high affinity to two receptors i.e., type-I and type-II IL-4 receptors. The type I IL-4 receptor consists of the IL-4 receptor α chain and the common γ C chain. The Type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain. IL-13 binds to the type-II IL-4 receptor, and to a unique receptor designated IL-13 receptor α2. The binding of IL-13 to the IL-13 receptor α2 does not transduce a signal and this receptor is also secreted in a soluble form. Accordingly, the IL-13 receptor α2 has often been referred to as a decoy receptor. Although IL-4, IL-13, IL-22, and IL-31, are critical cytokines for the development of immune responses that are required for protection against extracellular pathogens (e.g., tissue or lumen dwelling parasites), these cytokines also have been implicated in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

Atopic dermatitis (AD) is a relapsing pruritic and chronic inflammatory skin disease, that is characterized by immune system dysregulation and epidermal barrier abnormalities in humans. The pathological and immunological attributes of atopic dermatitis have been the subject of extensive investigations [reviewed in Rahman et al. Inflammation & Allergy-drug target 10:486-496 (2011) and Harskamp et al., Seminar in Cutaneous Medicine and Surgery 32:132-139 (2013)]. Atopic dermatitis is also a common condition in companion animals, especially dogs, where its prevalence has been estimated to be approximately 10-15% of the canine population. The pathogenesis of atopic dermatitis in dogs and cats [reviewed in Nuttall et al., Veterinary Records 172(8):201-207 (2013)] shows significant similarities to that of atopic dermatitis in man including skin infiltration by a variety of immune cells and CD4⁺ Th2 polarized cytokine milieu including the preponderance of IL-4, IL-13, and IL-31. In addition, IL-22 has been implicated in the exaggerated epithelial proliferation leading to epidermal hyperplasia that is characteristic of atopic dermatitis.

For example, antibodies against canine IL-31 have been shown to have a significant effect on pruritus associated with atopic dermatitis in dogs [U.S. Pat. No. 8,790,651 B2; U.S. Pat. No. 10,093,731 B2]. In addition, an antibody against human IL-31 receptor alpha (IL-31RA) has been tested and found to have a significant effect on pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New England Journal of Medicine, 376(9), 826-835 (2017)]. Accordingly, blocking IL-31 binding to its receptor IL-31RA, results in the relief of pruritus associated with atopic dermatitis.

Monoclonal antibodies raised against human IL-4 receptor alpha (IL-4 R_(α)) have been developed and some of these antibodies have been extensively tested for their therapeutic effects for treating atopic dermatitis in humans [see, e.g., US2015/0017176 A1]. More recently, caninized antibodies to canine IL-4 R_(α) that block the binding of canine IL-4 to canine IL-4 R_(α) also have been disclosed [US2018/0346580A1, hereby incorporated by reference in its entirety]. Because the Type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain, antibodies to canine IL-4 R_(α) have been obtained that can block both canine IL-4 and canine IL-13 from binding the Type II canine IL-4 receptor, thereby serving to help block the inflammation associated with atopic dermatitis [US2018/0346580A1].

Interleukin-22 (IL-22), also known as IL-10-related T cell-derived inducible factor (IL-TIF), belongs to the IL-10 cytokine family. IL-22 is produced by normal T cells upon anti-CD3 stimulation in humans. Mouse IL-22 expression is also induced in various organs upon lipopolysaccharide injection, suggesting that IL-22 may be involved in inflammatory responses. IL-22 binds specifically to, and signals through, a receptor complex consisting of a heterodimeric complex of IL-10R2 (also known as IL-10R beta) and the Interleukin-22 receptor (IL-22R) [see, Lee et al., Pharmacology Research & Perspectives, Pages 1-13 (2018:e00434)]. The Interleukin-22 receptor is also known as Interleukin-22R, alpha 1; IL-22RA1; IL-22R1, zcytor11; and CRF2-9 [Xu et at, Proc. Nat. Acad. Sci. 98 (17) 9511-9516 (2001); Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12): 1106-1110 (2010)]. IL-22 induces epithelial cell proliferation during wound healing, and its deficiency can enable uncontrolled proliferation and enhance tumor development [Huber et al., Nature 491:2.59-263 (2012]. IL-22 has been shown to activate STAT-1 and STAT-3 in several hepatoma cell lines and upregulate the production of acute phase proteins. Antibodies to Interleukin-22 and IL-22R act as anti-proliferative agents by blocking the interaction of IL-22 with IL-22R and thereby the related signaling pathway that leads to the epithelial proliferation.

However, despite recent success in treating atopic dermatitis, none of the current therapies employed result in a rapid onset of antipruritic action concomitant with a significant effect on the skin inflammation with an improvement in skin barrier function. Therefore, there is a need to design better therapies that can address one or more of the symptoms of atopic dermatitis.

The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.

SUMMARY OF THE INVENTION

The present invention provides new caninized antibodies to canine IL-4R alpha (IL-4R_(α)), which in particular embodiments are isolated, that have superior properties than those in the prior art, e.g., binding more tightly than prior art anti-canine IL-4 receptor alpha antibodies. In particular embodiments, the present invention provides mammalian antibodies or antigen binding fragments thereof that bind the canine interleukin-4 receptor alpha with specificity comprising a heavy chain that comprises a set of three heavy chain complementary determining regions (CDRs), a CDR heavy 1 (HCDR1), a CDR heavy 2 (HCDR2), and a CDR heavy 3 (HCDR3) in which the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 28. In other embodiments, the present invention provides mammalian antibodies or antigen binding fragments thereof that bind the canine interleukin-4 receptor alpha with specificity comprising a heavy chain that comprises a set of three heavy CDRs, HCDR1, HCDR2, and a HCDR3, in which the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49.

In related embodiments the mammalian antibodies or antigen binding fragments thereof that bind the canine interleukin-4 receptor α (IL-4R_(α)) with specificity further comprise a light chain that comprises a set of three light chain CDRs: a CDR light 1 (LCDR1), a CDR light 2 (LCDR2), and a CDR light 3 (LCDR3), in which the LCDR1 comprises the amino acid sequence of SEQ ID NO: 30, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 34. In preferred embodiments the mammalian antibody or antigen binding fragment thereof bind canine IL-4R_(α) and block the binding of canine IL-4R_(α) to canine interleukin-4 (cIL-4). In related embodiments the mammalian antibody or antigen binding fragment thereof bind canine IL-4R_(α) and block the binding of canine IL-4R_(α) to canine interleukin-13 (cIL-13). In still other embodiments, the mammalian antibody or antigen binding fragment thereof bind canine IL-4R_(α) and block the binding of canine IL-4R_(α) to cIL-4 and to cIL-13.

In specific embodiments the mammalian antibody to canine IL-4R alpha is a murine antibody. In related embodiments, the mammalian antibody to canine IL-4R alpha is a caninized murine antibody. In particular embodiments, the caninized antibody comprises a heavy chain that comprises an IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 6. In other embodiments, the caninized antibody comprises a heavy chain that comprises an IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 7. In still other embodiments, the caninized antibody comprises a heavy chain that comprises an IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 8. In yet other embodiments, the caninized antibody comprises a heavy chain that comprises an IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 9.

In specific embodiments, the caninized antibody comprises a heavy chain comprising a modified canine IgG-B (IgG-Bm) that comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the caninized antibody comprises a heavy chain that comprises the amino acid sequence SEQ ID NO: 36. In other embodiments, the caninized antibody comprises a heavy chain that comprises the amino acid sequence SEQ ID NO: 37. In still other embodiments, the caninized antibody comprises a heavy chain that comprises the amino acid sequence SEQ ID NO: 38. In specific embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence SEQ ID NO: 35. In alternative embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence SEQ ID NO: 43.

In particular embodiments, the caninized antibodies or antigen binding fragments thereof bind to SEQ ID NO: 46. In more particular embodiments, the caninized antibodies or antigen binding fragments thereof bind to one, two, three, four, five, six, or all seven of the following amino acid residues of canine IL-4R_(α): S₁₁₁, H₁₁₂, T₁₁₃, T₁₁₉, Y₁₂₂, T₁₂₄, and H₁₂₇ of SEQ ID NO: 5. In related embodiments, the caninized antibodies or antigen binding fragments thereof bind to SEQ ID NO: 47. In more particular embodiments, the caninized antibodies or antigen binding fragments thereof bind to one, two, three, four, five, six, seven, eight, nine, ten or all eleven of the following amino acid residues of canine IL-4R_(α): Y₁₅₀, T₁₅₃, Y₁₅₄, T₁₅₈, R₁₆₀, S₁₆₄, T₁₆₅, S₁₆₈, S₁₇₁, Y₁₇₂, S₁₇₃ of SEQ ID NO: 5. In still more particular embodiments, the caninized antibodies or antigen binding fragments thereof bind to both SEQ ID NO: 46 and SEQ ID NO: 47. In yet more particular embodiments of this type, the caninized antibodies or antigen binding fragments thereof bind to one, two, three, four, five, six, or all seven of the following amino acid residues of canine S₁₁₁, H₁₁₂, T₁₁₃, T₁₁₉, Y₁₂₂, T₁₂₄, and H₁₂₇ of SEQ ID NO: 5 and/or to one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following amino acid residues of canine IL-4R_(α): Y₁₅₀, T₁₅₃, Y₁₅₄, T₁₅₈, R₁₆₀, S₁₆₄, T₁₆₅, S₁₆₈, S₁₇₁, Y₁₇₂, S₁₇₃ of SEQ ID NO: 5.

The present invention also provides nucleic acids, including isolated nucleic acids, that encode the CDRs, the heavy chains of the caninized antibodies or antigen binding fragments thereof, and/or the light chains of the caninized antibodies or antigen binding fragments thereof. In addition, the present invention provides expression vectors that comprise such nucleic acids, and host cells that comprise such expression vectors.

In addition, the present invention provides pharmaceutical compositions that comprise the caninized antibodies and antigen binding fragments thereof of the present invention along with a pharmaceutically acceptable carrier and/or diluent. The present invention further provides methods of treating atopic dermatitis comprising administering one of the aforesaid compositions to a canine that has atopic dermatitis. In particular embodiments, the present invention provides methods of aiding in the blocking of inflammation associated with atopic dermatitis, comprising administering to a canine in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention.

These and other aspects of the present invention will be better appreciated by reference to the following Brief Description of the Drawings and the Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibition of IL-4-mediated STAT-6 phosphorylation by IL-4R alpha (IL-4R_(α)) antibodies. Two different caninized monoclonal anti-canine IL-4R_(α) antibodies designated c4H3 [see, WO2016/156588] and c152H11-H3L3 were evaluated for their ability to inhibit STAT-6 phosphorylation. The data show that both antibodies result in a dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4. The IL-4 control in the absence of IL-4R alpha (IL-4R_(α)) antibodies is shown in the upper right-hand portion of the graph.

FIG. 2 is a graph showing the inhibition of IL-13-mediated STAT-6 phosphorylation by IL-4R alpha antibodies. Two different caninized monoclonal anti-canine IL-4R_(α) antibodies designated c4H3 [see, WO2016/156588] and c152H11-H3L3 were evaluated for their ability to inhibit STAT-6 phosphorylation. The data shows that both antibodies result in a dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13. The IL-13 control in the absence of IL-4R alpha (IL-4R_(α)) antibodies is shown in the upper right-hand portion of the graph.

FIG. 3 shows the binding of caninized anti-canine IL-4R_(α) antibodies containing either lambda or kappa light chains as evaluated by ELISA. The results show that caninized anti-canine IL-4R_(α) antibodies containing lambda light chains (c152C1L1-H1, c152C1L1-H2 and c152C1L1-H3) bind to canine IL-4R_(α) as well as caninized anti-canine IL-4R_(α) antibodies containing the same CDRs, but with a kappa light chain (c152H11-H3L3). 152 mc is the mouse-canine chimeric antibody positive control and Iso-Ctr, the negative control, is an unrelated caninized antibody.

FIG. 4 shows the epitope comprising amino acid sequences SEQ ID NO: 46 and SEQ ID NO: 47 on canine IL-4R_(α) for the c152H11-H3L3 antibody.

DETAILED DESCRIPTION OF THE INVENTION

In response to need for better therapies for atopic dermatitis, the present invention provides caninized antibodies, formulations with the caninized antibodies, and methodologies that can achieve a significant effect on the skin inflammation associated with atopic dermatitis.

ABBREVIATIONS

Throughout the detailed description and examples of the invention the following abbreviations will be used:

ADCC Antibody-dependent cellular cytotoxicity CDC Complement-dependent cyotoxicity CDR Complementarity determining region in the immunoglobulin variable regions, defined using the Kabat numbering system EC50 concentration resulting in 50% efficacy or binding ELISA Enzyme-linked immunosorbant assay FR Antibody framework region: the immunoglobulin variable regions excluding the CDR regions. IC50 concentration resulting in 50% inhibition IgG Immunoglobulin G Kabat An immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb) V region The segment of IgG chains which is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain. VH Immunoglobulin heavy chain variable region VL Immunoglobulin light chain variable region VK Immunoglobulin kappa light chain variable region

Definitions

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

“Administration” and “treatment”, as it applies to an animal, e.g., a canine subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal e.g., a canine subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.

“Administration” and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term “subject” includes any organism, preferably an animal, more preferably a mammal (e.g., canine, feline, or human) and most preferably a canine.

“Treat” or “treating” means to administer a therapeutic agent, such as a composition containing any of the antibodies of the present invention, internally or externally to e.g., a canine subject or patient having one or more symptoms, or being suspected of having a condition, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate and/or ameliorate one or more disease/condition symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease/condition symptom (also referred to as the “therapeutically effective amount”) may vary according to factors such as the disease/condition state, age, and weight of the patient (e.g., canine), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease/condition symptom has been alleviated or ameliorated can be assessed by any clinical measurement typically used by veterinarians or other skilled healthcare providers to assess the severity or progression status of that symptom. While an embodiment of the present invention (e.g., a treatment method or article of manufacture) may not be effective in alleviating the target disease/condition symptom(s) in every subject, it should alleviate the target disease/condition symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the chi²-test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.

“Treatment,” as it applies to a human, veterinary (e.g., canine), or research subject, refers to therapeutic treatment, as well as research and diagnostic applications. “Treatment” as it applies to a human, veterinary (e.g., canine), or research subject, or cell, tissue, or organ, encompasses contact of the antibodies of the present invention to e.g., a canine or other animal subject, a cell, tissue, physiological compartment, or physiological fluid.

As used herein, the term “canine” includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

As used herein, the term “feline” refers to any member of the Felidae family. Members of this family include wild, zoo, and domestic members, including domestic cats, pure-bred and/or mongrel companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

As used herein the term “canine frame” refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues defined herein as CDR residues. With regard to a caninized antibody, in the majority of embodiments the amino acid sequences of the native canine CDRs are replaced with the corresponding foreign CDRs (e.g., those from a mouse antibody) in both chains. Optionally the heavy and/or light chains of the canine antibody may contain some foreign non-CDR residues, e.g., so as to preserve the conformation of the foreign CDRs within the canine antibody, and/or to modify the Fc function, as exemplified below and/or disclosed in U.S. Pat. No. 10,106,607 B2, hereby incorporated by reference herein in its entirety.

The “Fragment crystallizable region” abbreviated as “Fc” corresponds to the CH3-CH2 portion of an antibody that interacts with cell surface receptors called Fc receptors. The canine fragment crystallizable region (cFc) of each of the four canine IgGs were first described by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001); see also, Bergeron et al., Vet. Immunol. Immunopathol. 157: 31-41 (2014) and U.S. Pat. No. 10,106,607 B2].

As used herein the canine Fc (cFc) “IgG-Bm” is canine IgG-B Fc comprising two (2) amino acid residue substitutions, D31A and N63A in the amino acid sequence of SEQ ID NO: 10 of IgG-B (see below) and without the c-terminal lysine (′K″). Both the aspartic acid residue (D) at position 31 of SEQ ID NO: 10 and the asparagine residue (N) at position 63 of SEQ ID NO: 10, are substituted by an alanine residue (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly diminish the antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the naturally occurring canine IgG-B [see, U.S. Pat. No. 10,106,607 B2, the contents of which are hereby incorporated by reference in their entirety]. Further amino acid substitutions to the IgG-Bm are also envisioned, which parallel those which can be made in IgG-B and may include amino acid substitutions to favor heterodimer formation in bispecific antibodies. The amino acid sequence of IgG-B, SEQ ID NO: 45 is:

1                                                   50 LGGPSVFIFP PKPKDTLLIA RTPEVTCVVV  D LDPEPPEVQ ISWFVDGKQM

 CH2 51                                                 100 QTAKTQPREE QF N GTYRVVS VLPIGHQDWL KGKQFTCKVN NKALPSPIER 101                                                150 TISKARGQAH QPSVYVLPPS REELSKNTVS LTCLIKDFFP PDIDVEWQSN        

 CH3 151                                                200 GQQEPESKYR TTPPQLDEDG SYFLYSKLSV DKSRWQRGDT FICAVMHEAL 210          215 HNHYTQKSLS HSPGK

The amino acid sequence of IgG-Bm, SEQ ID NO: 10, is provided below.

LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWEVDGKQM QTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER TISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSN GQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTOESLSHSPG

As used herein, a “substitution of an amino acid residue” with another amino acid residue in an amino acid sequence of an antibody for example, is equivalent to “replacing an amino acid residue” with another amino acid residue and denotes that a particular amino acid residue at a specific position in the amino acid sequence has been replaced by (or substituted for) by a different amino acid residue. Such substitutions can be particularly designed i.e., purposefully replacing an alanine with a serine at a specific position in the amino acid sequence by e.g., recombinant DNA technology. Alternatively, a particular amino acid residue or string of amino acid residues of an antibody can be replaced by one or more amino acid residues through more natural selection processes e.g., based on the ability of the antibody produced by a cell to bind to a given region on that antigen, e.g., one containing an epitope or a portion thereof, and/or for the antibody to comprise a particular CDR that retains the same canonical structure as the CDR it is replacing. Such substitutions/replacements can lead to “variant” CDRs and/or variant antibodies.

As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. An antibody can be a monomer, dimer, or larger multimer. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies and camelized single domain antibodies. “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as caninization of an antibody for use as a canine therapeutic antibody.

As used herein, antibodies of the present invention that “block” or is “blocking” or is “blocking the binding” of e.g., a canine receptor to its binding partner (ligand), is an antibody that blocks (partially or fully) the binding of the canine receptor to its canine ligand and vice versa, as determined in standard binding assays (e.g., BIACore®, ELISA, or flow cytometry).

Typically, an antibody or antigen binding fragment of the invention retains at least 10% of its canine antigen binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine antigen binding affinity as the parental antibody. It is also intended that an antibody or antigen binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.

“Isolated antibody” refers to the purification status and in such context means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with experimental or therapeutic use of the binding compound as described herein.

As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. [U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)]. Typically the variable domains are obtained from an antibody from an experimental animal (the “parental antibody”), such as a rodent, and the constant domain sequences are obtained from the animal subject antibodies, e.g., human or canine so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human or canine subject respectively, than the parental (e.g., rodent) antibody.

As used herein, the term “caninized antibody” refers to forms of antibodies that contain sequences from both canine and non-canine (e.g., murine) antibodies. In general, the caninized antibody will comprise substantially all of at least one or more typically, two variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising 6 CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) are those of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody comprises both the three heavy chain CDRs and the three light chain CDRS from a murine anti-canine antigen antibody together with a canine frame or a modified canine frame. A modified canine frame comprises one or more amino acids changes as exemplified herein that further optimize the effectiveness of the caninized antibody, e.g., to increase its binding to its canine antigen and/or its ability to block the binding of that canine antigen to the canine antigen's natural binding partner.

The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same. Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e. LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain). [See Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.

Public Health Service, National Institutes of Health, Bethesda, Md. (1991), defining the CDR regions of an antibody by sequence; see also Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) defining the CDR regions of an antibody by structure]. As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

There are four known IgG heavy chain subtypes of dog IgG and they are referred to as IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are referred to as lambda and kappa. In specific embodiments of the invention, besides binding and activating of canine immune cells, a canine or caninized antibody against its antigen of the present invention optimally has two attributes:

1. Lack of effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

2. be readily purified on a large scale using industry standard technologies such as that based on protein A chromatography.

None of the naturally occurring canine IgG isotypes satisfy both criteria. For example, IgG-B can be purified using protein A, but has high level of ADCC activity. On the other hand, IgG-A binds weakly to protein A, but also displays ADCC activity. Moreover, neither IgG-C nor IgG-D can be purified on protein A columns, although IgG-D displays no ADCC activity. (IgG-C has considerable ADCC activity). One way the present invention addresses these issues is by providing modified canine IgG-B antibodies of the present invention specific to an antigen of the present invention that lack the effector functions such as ADCC and can be easily purified using industry standard protein A chromatography.

As used herein an “anti-inflammatory antibody” is an antibody that can act as an anti-inflammatory agent in an animal, including a mammal such as a human, a canine, and/or a feline, particularly with respect to atopic dermatitis. In particular embodiments, the anti-inflammatory antibody binds to specific proteins in the IL-4/IL-13 signaling pathway, such as IL-4 or the receptor IL-4R_(α). The binding of the anti-inflammatory antibody to its corresponding antigen (e.g., IL-4 or IL-4R_(α)) inhibits the binding of e.g., IL-4 with IL-4R_(α), and interferes with and/or prevents the signaling of this pathway, thereby interfering with or preventing the chronic inflammation associated with atopic dermatitis.

“Homology”, as used herein, refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are optimally aligned. When a position in both of the two compared sequences is occupied by the same base or amino acid residue, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared ×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous when the sequences are optimally aligned then the two sequences are 60% homologous. Generally, the comparison is made when two sequences are aligned to give maximum percent homology. Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.

As used herein one amino acid sequence is 100% “identical” to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% “identical” to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In particular embodiments, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account. Sequence similarity includes identical residues and nonidentical, biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable.

“Conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table A directly below.

TABLE A Exemplary Conservative Amino Acid Substitutions Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gin Gly (G) Ala His (H) Asn; Gin Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro (P) Ala; Gly Ser(S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr(Y) Trp; Phe Val (V) Ile; Leu

Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. “Function-conservative variants,” as used herein, refers to antibodies or fragments in which one or more amino acid residues have been changed without altering a desired property, such an antigen affinity and/or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table A above.

“Isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

The present invention provides isolated caninized antibodies of the present invention, methods of use of the antibodies in the treatment of a condition e.g., the treatment of atopic dermatitis in canines. In canine, there are four IgG heavy chains referred to as A, B, C, and D. These heavy chains represent four different subclasses of dog IgG, which are referred to as IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC) and IgG-D (or IgGD). Each of the two heavy chains consists of one variable domain (VH) and three constant domains referred to as CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain via an amino acid sequence referred to as the “hinge” or alternatively as the “hinge region”.

The nucleic acid and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. The amino acid and nucleic sequences for these heavy chains are also available from the GenBank data bases. For example, the amino acid sequence of IgGA heavy chain has accession number AAL35301.1, IgGB has accession number AAL35302.1, IgGC has accession number AAL35303.1, and IgGD has accession number (AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequence of these light chains can be obtained from GenBank Databases. For example, the kappa light chain amino acid sequence has accession number ABY 57289.1 and the lambda light chain has accession number ABY 55569.1.

In the present invention, the amino acid sequence for each of the four canine IgG Fc fragments is based on the identified boundary of CH1 and CH2 domains as determined by Tang et al, supra. Caninized murine anti-canine antibodies that bind canine IL-4R_(α) include, but are not limited to: antibodies of the present invention that comprise canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and/or canine kappa or lambda light chains together with murine anti-canine IL-4R_(α) CDRs. Accordingly, the present invention provides isolated caninized murine anti-canine antibodies of the present invention that bind to canine IL-4R_(α) and block the binding of that canine IL-4R_(α) to their natural binding partners canine IL-4 and/or canine IL-13.

Accordingly, the present invention further provides caninized murine antibodies and methods of use of the antibodies of the present invention in the treatment of a condition e.g., the treatment of atopic dermatitis in canines.

The present invention further provides full length canine heavy chains that can be matched with corresponding light chains to make a caninized antibody. Accordingly, the present invention further provides caninized murine anti-canine antigen antibodies (including isolated caninized murine anti-canine antibodies) of the present invention and methods of use of the antibodies of the present invention in the treatment of a condition e.g., the treatment of atopic dermatitis in canines.

The present invention also provides antibodies of the present invention that comprise a canine fragment crystallizable region (cFc region) in which the cFc has been genetically modified to augment, decrease, or eliminate one or more effector functions. In one aspect of the present invention, the genetically modified cFc decreases or eliminates one or more effector functions. In another aspect of the invention the genetically modified cFc augments one or more effector function. In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a particular embodiment the effector function is antibody-dependent cytotoxicity (ADCC) that is augmented, decreased, or eliminated. In another embodiment the effector function is complement-dependent cytotoxicity (CDC) that is augmented, decreased, or eliminated. In yet another embodiment, the cFc region has been genetically modified to augment, decrease, or eliminate both the ADCC and the CDC.

In order to generate variants of canine IgG that lack effector functions, a number of mutant canine IgGB heavy chains were generated. These variants may include one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. Variant heavy chains (i.e., containing such amino acid substitutions) were cloned into expression plasmids and transfected into HEK 293 cells along with a plasmid containing the gene encoding a light chain. Intact antibodies expressed and purified from HEK 293 cells were evaluated for binding to Fc_(γ)RI and C1q to assess their potential for mediation of immune effector functions. [See, U.S. Pat. No. 10,106,607 B2, the contents of which are hereby incorporated by reference in its entirety.]

The present invention also provides modified canine IgG-Ds which in place of its natural IgG-D hinge region they comprise a hinge region from:

IgG-A: SEQ ID NO: 6 FNECRCTDTPPCPVPEP IgG-B: SEQ ID NO: 7 PKRENGRVPRPPDCPKCPAPEM; or IgG-C: SEQ ID NO: 8 AKECECKCNCNNCPCPGCGL.

Alternatively, the IgG-D hinge region can be genetically modified by replacing a serine residue with a proline residue, i.e., PKESTCKCIPPCPVPES, SEQ ID NO: 9 (with the proline residue (P) underlined and in bold substituting for the naturally occurring serine residue). Such modifications can lead to a canine IgG-D lacking fab arm exchange. The modified canine IgG-Ds can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. In order to construct these variants, the nucleic acids encoding the amino acid sequence of canine IgG-D can be modified so that it encodes the modified IgG-Ds. The modified nucleic acid sequences are then cloned into expression plasmids for protein expression.

The six complementary determining regions (CDRs) of a caninized murine anti-canine antibody, as described herein can comprises a canine antibody kappa light chain comprising a murine light chain LCDR1, LCDR2 and LCDR3 and a canine antibody heavy chain IgG comprising a murine heavy chain HCDR1, HCDR2 and HCDR3.

Nucleic Acids

The present invention further comprises the nucleic acids encoding the antibodies of the present invention (see e.g., Examples below).

Also included in the present invention are nucleic acids that encode immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of the caninized antibodies, with the exception of the CDRs which do not change, provided herein when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides nucleic acids that encode immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when the comparison is performed with a BLAST algorithm, wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.

As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program using the default parameters.

The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141(1996); Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J. M. et al., Comput. Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3.” (1978), M. O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S. F., J. Mol. Biol. 219:555-565 (1991); States, D. J., et al., Methods 3:66-70(1991); Henikoff, S., et al., Proc. Natl. Acad Sci. USA 89:10915-10919 (1992); Altschul, S. F., et al., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S., et al., Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990); Karlin, S., et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

Antibodies of the present invention can be produced recombinantly by methods that are known in the field. Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and/or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, comprise the instant invention, independent of the glycosylation pattern that the antibodies may have. Similarly, in particular embodiments, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo [See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775].

Canine IL-4 Receptor Alpha Receptor

The cDNA encoding a predicted full length canine IL-4 receptor alpha chain (SEQ ID NO: 1) was identified through a search of the Genbank database (accession #XM_547077.4; see also, U.S. Pat. No. 7,208,579 B2). This predicted cDNA encodes 823 amino acids (SEQ ID NO: 2) including a 25 amino acid leader sequence and is identified as accession #XP_547077.3. The mature predicted canine IL-4 receptor α chain protein (SEQ ID NO: 4) shares 65% identity with human IL-4 receptor α chain (accession #NP_000409.1) and 70% identity with swine IL-4 receptor a chain (accession #NP_999505.1). The mature predicted canine IL-4 receptor α chain protein is encoded by the nucleotide sequence identified as SEQ ID NO: 3. Comparison of the predicted mature IL-4 receptor α chain with the known sequences of human IL-4 receptor α chain identified the extracellular domain (ECD) of the mature canine IL-4 receptor α chain protein and is designated as SEQ ID NO: 5. This has all been previously described in [US2018/0346580; hereby incorporated herein in its entirety].

Canine IL-4 receptor a chain full length DNA with signal sequence [SEQ ID NO: 1] atgggcagactgtgcagcggcctgaccttccccgtgagctgcctggtgctggtgtgggtggccagcagcggcagcgt gaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccacccca ccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgag aacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacct gtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacc tgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcac agcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacat gggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggccc agacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcac ctgcccctgggcgtgagcatcagctgcctggtgatcctggccatctgcctgagctgctacttcagcatcatcaagat caagaagggctggtgggaccagatccccaaccccgcccacagccccctggtggccatcgtgatccaggacagccagg tgagcctgtggggcaagagaagcagaggccaggagcccgccaagtgcccccactggaagacctgcctgaccaagctg ctgccctgcctgctggagcacggcctgggcagagaggaggagagccccaagaccgccaagaacggccccctgcaggg ccccggcaagcccgcctggtgccccgtggaggtgagcaagaccatcctgtggcccgagagcatcagcgtggtgcagt gcgtggagctgagcgaggcccccgtggacaacgaggaggaggaggaggtggaggaggacaagagaagcctgtgcccc agcctggagggcagcggcggcagcttccaggagggcagagagggcatcgtggccagactgaccgagagcctgttcct ggacctgctgggcggcgagaacggcggcttctgcccccagggcctggaggagagctgcctgcccccccccagcggca gcgtgggcgcccagatgccctgggcccagttccccagagccggccccagagccgcccccgagggccccgagcagccc agaagacccgagagcgccctgcaggccagccccacccagagcgccggcagcagcgccttccccgagcccccccccgt ggtgaccgacaaccccgcctacagaagcttcggcagcttcctgggccagagcagcgaccccggcgacggcgacagcg accccgagctggccgacagacccggcgaggccgaccccggcatccccagcgccccccagccccccgagccccccgcc gccctgcagcccgagcccgagagctgggagcagatcctgagacagagcgtgctgcagcacagagccgcccccgcccc cggccccggccccggcagcggctacagagagttcacctgcgccgtgaagcagggcagcgcccccgacgccggcggcc ccggcttcggccccagcggcgaggccggctacaaggccttctgcagcctgctgcccggcggcgccacctgccccggc accagcggcggcgaggccggcagcggcgagggcggctacaagcccttccagagcctgacccccggctgccccggcgc ccccacccccgtgcccgtgcccctgttcaccttcggcctggacaccgagccccccggcagcccccaggacagcctgg gcgccggcagcagccccgagcacctgggcgtggagcccgccggcaaggaggaggacagcagaaagaccctgctggcc cccgagcaggccaccgaccccctgagagacgacctggccagcagcatcgtgtacagcgccctgacctgccacctgtg cggccacctgaagcagtggcacgaccaggaggagagaggcaaggcccacatcgtgcccagcccctgctgcggctgct gctgcggcgacagaagcagcctgctgctgagccccctgagagcccccaacgtgctgcccggcggcgtgctgctggag gccagcctgagccccgccagcctggtgcccagcggcgtgagcaaggagggcaagagcagccccttcagccagcccgc cagcagcagcgcccagagcagcagccagacccccaagaagctggccgtgctgagcaccgagcccacctgcatgagcg ccagc Canine IL-4 receptor a full length protein with signal sequence in bold font [SEQ ID NO: 2] MGRLCSGLTFPVSCLVLVWVASSGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPE NREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLH SELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQH LPLGVSISCLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKL LPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCP SLEGSGGSFQEGREGIVARLTESLFLDLLGGENGGFCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQP RRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSFGSFLGQSSDPGDGDSDPELADRPGEADPGIPSAPQPPEPPA ALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPG TSGGEAGSGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLA PEQATDPLRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLE ASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSAS Canine IL-4 receptor a mature full length protein without signal sequence [SEQ ID NO: 4] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLD LWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTY MGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLPLGVSISCLVILAICLSCYFSIIK IKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGREEESPKTAKNGPLQ GPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGREGIVARLTESLF LDLLGGENGGFCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRRPESALQASPTQSAGSSAFPEPPP VVTDNPAYRSFGSFLGQSSDPGDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHRAAPA PGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPGTSGGEAGSGEGGYKPFQSLTPGCPG APTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHL CGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQP ASSSAQSSSQTPKKLAVLSTEPTCMSAS Canine IL-4 receptor a mature full length DNA without signal sequence [SEQ ID NO: 3] gtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccc caccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccg agaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggac ctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaa cctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgc acagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctac atgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggc ccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagc acctgcccctgggcgtgagcatcagctgcctggtgatcctggccatctgcctgagctgctacttcagcatcatcaag atcaagaagggctggtgggaccagatccccaaccccgcccacagccccctggtggccatcgtgatccaggacagcca ggtgagcctgtggggcaagagaagcagaggccaggagcccgccaagtgcccccactggaagacctgcctgaccaagc tgctgccctgcctgctggagcacggcctgggcagagaggaggagagccccaagaccgccaagaacggccccctgcag ggccccggcaagcccgcctggtgccccgtggaggtgagcaagaccatcctgtggcccgagagcatcagcgtggtgca gtgcgtggagctgagcgaggcccccgtggacaacgaggaggaggaggaggtggaggaggacaagagaagcctgtgcc ccagcctggagggcagcggcggcagcttccaggagggcagagagggcatcgtggccagactgaccgagagcctgttc ctggacctgctgggcggcgagaacggcggcttctgcccccagggcctggaggagagctgcctgcccccccccagcgg cagcgtgggcgcccagatgccctgggcccagttccccagagccggccccagagccgcccccgagggccccgagcagc ccagaagacccgagagcgccctgcaggccagccccacccagagcgccggcagcagcgccttccccgagccccccccc gtggtgaccgacaaccccgcctacagaagcttcggcagcttcctgggccagagcagcgaccccggcgacggcgacag cgaccccgagctggccgacagacccggcgaggccgaccccggcatccccagcgccccccagccccccgagccccccg ccgccctgcagcccgagcccgagagctgggagcagatcctgagacagagcgtgetgcagcacagagccgcccccgcc cccggccccggccccggcagcggctacagagagttcacctgcgccgtgaagcagggcagcgcccccgacgccggcgg ccccggcttcggccccagcggcgaggccggctacaaggccttctgcagcctgctgcccggcggcgccacctgccccg gcaccagcggcggcgaggccggcagcggcgagggcggctacaagcccttccagagcctgacccccggctgccccggc gcccccacccccgtgcccgtgcccctgttcaccttcggcctggacaccgagccccccggcagcccccaggacagcct gggcgccggcagcagccccgagcacctgggcgtggagcccgccggcaaggaggaggacagcagaaagaccctgctgg cccccgagcaggccaccgaccccctgagagacgacctggccagcagcatcgtgtacagcgccctgacctgccacctg tgcggccacctgaagcagtggcacgaccaggaggagagaggcaaggcccacatcgtgcccagcccctgctgcggctg ctgctgcggcgacagaagcagcctgctgctgagccccctgagagcccccaacgtgctgcccggcggcgtgctgctgg aggccagcctgagccccgccagcctggtgcccagcggcgtgagcaaggagggcaagagcagccccttcagccagccc gccagcagcagcgcccagagcagcagccagacccccaagaagctggccgtgctgagcaccgagcccacctgcatgag egeeage Canine IL-4 receptor a chain extracellular domain [SEQ ID NO: 5] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLD LWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTY MGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLP

Antibody Protein Engineering

By way of example, and not limitation, the canine heavy chain constant region can be from IgG-B or a modified cFc, such as the IgG-Bm used herein [see, U.S. Pat. No. 10,106,607 B2, hereby incorporated by reference in its entirety] and the canine light chain constant region can be from kappa.

The antibodies can be engineered to include modifications to the canine framework and/or the canine frame residues within the variable domains of a parental (i.e., mouse) monoclonal antibody, e.g. to improve the properties of the antibody. In specific circumstances, an individual CDR can be modified, as exemplified below.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions comprising the antibodies of the present invention, these antibodies can be admixed with a pharmaceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)].

Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions [see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.]. In one embodiment, the antibodies of the present invention are diluted to an appropriate concentration in a sodium acetate solution pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD₅₀/ED₅₀). In particular aspects, antibodies exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in canines. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial. In particular embodiments, the antibodies of the present invention can be administered by an invasive route such as by injection. In further embodiments of the invention, the antibodies of the present invention, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector. The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules form administering pharmaceutical compositions include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

Alternatively, one may administer the antibodies of the present invention in a local rather than systemic manner, often in a depot or sustained release formulation.

The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibodies, the level of symptoms, the immunogenicity of the therapeutic antibodies and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibodies to effect improvement in the target disease/condition state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibodies and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available [see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y. (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y. (1993); Baert, et al. New Engl. J. Med 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J Med 344:783-792 (2001); Beniaminovitz et al. New Engl. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)].

Determination of the appropriate dose is made by the veterinarian, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of the symptoms.

Antibodies provided herein may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more [see, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52:133-144 (2003)]. Doses may also be provided to achieve a pre-determined target concentration of antibodies of the present invention in the canine's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more. In other embodiments, antibodies of the present invention is administered subcutaneously or intravenously, on a weekly, biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, “inhibit” or “treat” or “treatment” includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject (e.g., a canine) with a disorder, condition and/or symptom, or with the potential to develop such a disorder, disease or symptom.

As used herein, the terms “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to an amount of antibodies of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, e.g., canine, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the antibodies sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess severity of the condition.

EXAMPLES Example 1 Anti-IL-4 Receptor Alpha Antibodies

General Material and Methods

The recombinant proteins were obtained by providing the amino acid sequence for a selected protein to a commercial manufacturer (ATUM, Newark, Calif.), who in turn chose an appropriate nucleotide sequence that encoded this amino acid sequence. The nucleotide sequences can also be obtained from publicly available DNA databases, such as GenBank. The commercial manufacturer then chemically synthesized the nucleic acid, which next was cloned by ATUM into an expression plasmid (pD2610-v10; available from AUTM) for producing the corresponding recombinant protein. The plasmid was placed into either HEK-293 cells or CHO cells to express the recombinant protein, which was then isolated by conventional methods.

Balb/c mice were immunized multiple times (with 10 μg each time) over a 17 day period. The immunizing antigen was the canine IL-4 R alpha chain extracellular domain (ECD)-human Fc fusion protein. Following immunization, serum was collected from each mouse and tested for reactivity with canine IL-4 receptor alpha chain ECD HIS-tagged protein. The spleen cells of the mouse with the highest serum anti-IL-4 receptor alpha chain ECD titer were fused to the myeloma P3X63Ag8.653 cell line. Approximately 2 weeks following fusion, supernatant from putative hybridoma cells were tested by ELISA for their reactivity to the IL-4 receptor alpha chain ECD HIS-tagged protein. Hybridomas producing strong positive signals in the ELISA were subcloned by limiting dilution and tested again for reactivity to canine IL-4 receptor alpha chain ECD HIS-tagged protein. Anti-canine IL-4 receptor alpha antibodies include the antibodies c152H11VL3-cCLk-s/c152H11VH3-cIgG-Bm and the antibody c146E2VL3-cCLk-s/c146E2VH3-cIgG-Bm.

Sets of the six (6) CDRs (three individual light chains (LC) and three heavy chains (HC) sequences) for these two antibody families are provided below in Table 1A (nucleic acid sequences) and Table 1B (amino acid sequences). Table 1B includes a modified murine HCDR3 for 152H11 comprising the amino acid sequence of SEQ ID NO: 28, whereas the rest are unmodified murine CDRs. Table 1A provides nucleic acids that encode the twelve CDRs listed in Table 1B. Table 1C provides both the amino acid sequence (SEQ ID NO: 49) of the unmodified murine HCDR3 for 152H11 and a nucleic acid sequence (SEQ ID NO: 48) that encodes this unmodified murine HCDR3. The amino acid sequence (SEQ ID NO: 28) of the modified murine HCDR3 of the 152H11 antibody family differs from that of the corresponding unmodified murine HCDR3 by the replacement of the C-terminal cysteine residue of the amino acid sequence of SEQ ID NO: 49 with a serine residue. The unmodified murine HCDR3 (SEQ ID NO: 49) of the 152H11 antibody family is found in both c152H11VH1-cIgGBm (SEQ ID NO: 36) and c152H11VH2-cIgGBm (SEQ ID NO: 37), whereas c152H11VH3-cIgGBm (SEQ ID NO: 38) comprises the modified murine HCDR3 (SEQ ID NO: 28). The amino acid sequences of full length light chains and heavy chains for the caninized antibodies are provided immediately following Table 1C.

TABLE 1A IL-4 R alpha ANTIBODY CDR NUCLEIC ACID AND AMINO ACID SEQUENCES SEQ CDR ID NO: 146E2 HCDR1 agatactggatgcac 11 HCDR2 atgattcaccccgacagcggcaacatcaactacaacga 13 gcggttcaagacc HCDR3 cagctgcggaacgccatggattat 15 LCDR1 agagccagcgagagcgtggacagctacggcaacagctt 17 cctgaac LCDR2 agagccagcaacctggcctct 19 LCDR3 cagcagaactacgagaaccccagaacc 21 152H11 HCDR1 agetacggcatgage 23 HCDR2 acaatcagcagaggcggcgactacacctactatcccga 25 cagcgtgaagggc HCDR3 ^(#)ggcaccctgaacaaccggggctttgcttct 27 LCDR1 aaggccagccagaacgtgggcaccaatgtggcc 29 LCDR2 agcgccagctaccggtactct 31 LCDR3 cagcagtacaacagctacccctacacc 33 ^(#)See footnote for Table 1B below.

TABLE IB SEQ SEQ ID ID 146E2 NO: 152H11 NO: HCDR1 RYWMH 12 SYGMS 24 HCDR2 MIHPDSGNINYNERFKT 14 TISRGGDYTYYPDSVKG 26 HCDR3 QLRNAMDY 16 ^(#)GTLNNRGFAS 28 LCDR1 RASESVDSYGNSFLN 18 KASQNVGTNVA 30 LCDR2 RASNLAS 20 SASYRYS 32 LCDR3 QQNYENPRT 22 QQYNSYPYT 34 ^(#)The C-terminal cysteine residue of HCDR3 of 152H11 was replaced with a serine residue as discussed above.

TABLE 1C SEQ CDR 152H11 NA AA ID NO: HCDR3 ggcaccctgaacaaccgcggctttgcgtgc √ 48 HCDR3 GTLNNRGFAC √ 49

c152H11L3-cCLk-s (kappa light chain): [SEQ ID NO: 35] EIVMTQSPASLSLSQEEKVTITCKASQNVGTNVAWYQQKPGQAPKLLIY SASYRYSGLPDRFSGSGSGTDFSFTISSLEPEDVAEFFCQQYNSYPYTF GQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVK WKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEIT HKSLPSTLIKSFQRSECQRVD C152H11VH1-cIgGBm (heavy chain): [SEQ ID NO: 36] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLQWVA TISRGGDYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAK GTLNNRGFACWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACL VSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSV FIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKT QPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISK ARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQ EPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG c152HHVH2-cIgGBm (heavy chain): [SEQ ID NO: 37] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPDKRLQWVA TISRGGDYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAR GTLNNRGFACWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACL VSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSV FIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKT QPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISK ARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQ EPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG C152H11VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 38] EVQLVESGGDLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPDKRLQWVA TISRGGDYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAR GTLNNRGFASWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACL VSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSV FIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKT QPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISK ARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQ EPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG c146E2VL3-cCLk-s (kappa light chain): [SEQ ID NO: 39] DIVLTQTPLSLSVSPGETASIYCRASESVDSYGNSFLNWYQQKPGQPPK LLIYRASNLASEIPDRFSGSGSRTEFTLKISRVEADDAGVYYCQQNYEN PRTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKD INVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYS CEITHKSLPSTLIKSFQRSECQRVD c146E2VH1-cIgGBm (heavy chain): [SEQ ID NO: 40] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWVRQAPGAGLDWMG MIHPDSGNINYNERFKTRVTLTADTSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVS GYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSE TFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFI FPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQP REEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKAR GQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHY TQESLSHSPG c146E2VH2-cIgGBm (heavy chain): [SEQ ID NO: 41] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQAPGAGLDWIG MIHPDSGNINYNERFKTKATLTADTSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVS GYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSE TFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFI FPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQP REEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKAR GQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHY TQESLSHSPG c146E2VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 42] EVQLVQSGAEVKKPGASVKVSCKASGYTFARYWMHWMKQAPGAGLDWIG MIHPDSGNINYNERFKTKATLTVDKSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVS GYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSE TFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFI FPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQP REEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKAR GQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHY TQESLSHSPG In addition, the light chains for the canine IL-4 receptor alpha antibodies were also constructed with a lambda light chain as provided below. C152H11LVl-cC1 (lambda light chain) [SEQ ID NO: 43] QSVLTQPASVSGSLGQRVTISCKASQNVGTNVAWYQQLPGTSPRTLIYS ASYRYSGVPDRFSGSRSGSTATLTISGLQAEDEADYYCQQYNSYPYTFG GGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVA WKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVT HEGSTVEKKVAPAECS c146E2LV1-cC1 (lambda light chain) [SEQ ID NO: 44] QSVLTQPASVSGSLGQRVTISCRASESVDSYGNSFLNWYQQLPGKAPSL LIYRASNLASGVPERFSGSKSGSSATLTITGLQAEDEADYYCQQNYENP RTFGGGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSG VTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFS CLVTHEGSTVEKKVAPAECS

Example 2 STAT-6 Inhibition

Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 Cell as follows:

Materials

-   -   1. Actively growing DH82 cells     -   2. DH82 Cell Growth Media (ATCC® 302003™, Eagle's Minimum         Essential Medium supplied with heat-inactivated fetal bovine         serum to a final concentration of 15% w/v)     -   3. AlphaLISA p-STAT6 (Tyr641) Assay Kit: Perkin Elmer Catalog:         ALSU-PST6-A-HV     -   4. Recombinant canine IL-4: R&D Systems, Catalog: 752-CL/CF     -   5. Recombinant canine IL-13: R&D Systems, Catalog: 5894-CL/CF     -   6. Perkin Elmer Envision         -   a. Caninized anti-canine IL-4R_(α) monoclonal antibodies         -   b. c152H11-H3L3         -   c. 4H3 caninized antibodies from US2018/0346580

Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 Cell as follows:

Methods

-   -   1. Two tissue culture plates were seeded with 8×10⁴ DH82 cells         per well (200 μL with the density of 4×10⁵ cells/mL) and         incubated at 37° C. overnight.     -   2. The test antibodies were pre-diluted to 500 μg/mL and then         3-fold serially diluted in DH82 Cell Growth Media. The media was         removed from the cell culture plates and 50 μL/well of the         serially diluted test sample were transferred to each plate.     -   3. Canine IL-4 was diluted to 5 ng/mL in DH82 Cell Growth Media         and 50 μL was added to each well of one plate. Canine IL-13 was         diluted to 10 ng/mL in DH82 Cell Growth Media and 50 μL was         added to each well of the second plate. The plates were         incubated for 15 min at 37° C.     -   4. The media was removed from the plates and 100 μL per well of         freshly prepared 1× lysis buffer from the AlphaLISA p-STAT-6         Assay Kit was added to the plate. The plate was agitated on a         plate shaker with 350 rpm for 10 minutes at room temperature.     -   5. The Acceptor Mix was prepared from the AlphaLISA p-STAT6         Assay Kit and 15 per well was added to 30 μL of the cell lysate         in 96-well ½ Area Plates. The plates were sealed, agitated for 2         minutes at 350 rpm, and then incubated for 2 hours at room         temperature.     -   6. The Donor Mix was prepared from the AlphaLISA p-STAT6 Assay         kit under subdued laboratory lighting and 15 μL per well was         added to each plate. The plates were sealed, covered with foil,         agitated for 2 minutes at 350 rpm, and then incubated for 2         hours at room temperature.     -   7. The plates were read using the AlphaScreen settings on the         Perkin Elmer EnVison.

Two different caninized monoclonal anti-canine IL-4R_(α) antibodies designated c4H3 [WO2016/156588; US2018/0346580] and c152H11-H3L3 were evaluated for their ability to inhibit αSTAT-6 phosphorylation by blocking the binding of either canine IL-4 or canine IL-13 to canine IL-4R_(α). The data shown in FIG. 1 demonstrate that both antibodies result in a dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4, but surprisingly, c152H11-H3L3 bound more tightly than the prior art anti-canine IL-4 receptor alpha antibody c4H3 [WO2016/156588]. The IL-4 control in the absence of IL-4R alpha (IL-4R_(α)) antibodies is shown in the upper right-hand portion of the graph. The data shown in FIG. 2 also demonstrates that that both antibodies result in a dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13, with c152H11-H3L3 again binding more tightly than the prior art anti-canine IL-4 receptor alpha antibody c4H3. The IL-13 control in the absence of IL-4R alpha (IL-4R_(α)) antibodies is shown in the upper right hand portion of the graph. FIG. 3 shows that replacing the kappa light chain with the lambda light chain had no effect on the binding of c152H11-H3L3 to IL-4R_(α).

Example 3 Epitope Mapping

The interaction of antibodies with their cognate protein antigens is mediated through the binding of specific amino acids of the antibodies (paratopes) with specific amino acids (epitopes) of target antigens. An epitope is an antigenic determinant that causes a specific reaction by an immunoglobulin. An epitope consists of a group of amino acids on the surface of the antigen. A protein of interest may contain several epitopes that are recognized by different antibodies. The epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by a stretch of a continuous sequence of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence, but are brought together upon three-dimensional protein folding.

Epitope mapping refers to the process of identifying the amino acid sequences (i.e., epitopes) that are recognized by antibodies on their target antigens. Identification of epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it can aid in the development of new therapeutics, diagnostics, and vaccines. Epitope mapping can also aid in the selection of optimized therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on IL-4 receptor alpha can also elucidate unique epitopes, and define the protective or pathogenic effects of vaccines. Epitope identification also can lead to development of subunit vaccines based on chemical or genetic coupling of the identified peptide epitope to a carrier protein or other immunostimulating agents.

Epitope mapping can be carried out using polyclonal or monoclonal antibodies and several methods are employed for epitope identification depending on the suspected nature of the epitope (i.e., linear versus conformational). Mapping linear epitopes is more straightforward and relatively, easier to perform. For this purpose, commercial services for linear epitope mapping often employ peptide scanning. In this case, an overlapping set of short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind antibodies of interest. The strategy is rapid, high-throughput, and relatively inexpensive to perform. On the other hand, mapping of a discontinuous epitope is more technically challenging and requires more specialized techniques such as x-ray co-crystallography of a monoclonal antibody together with its target protein, Hydrogen-Deuterium (H/D) exchange, Mass Spectrometry coupled with enzymatic digestion as well as several other methods known to those skilled in the art.

Mapping of Canine IL-4 Receptor Alpha Epitopes Using Mass Spectroscopy:

A method based on chemical crosslinking, mass spectrometry detection, and covalent tagging as employed to identify epitopes recognized by anti-canine IL-4 receptor alpha mAbs [CovalX Instruments Incorporated located at 999 Broadway, Suite 305, Saugus, Mass. 01906-4510 USA).]

The application of this technology to epitope mapping of canine IL-4 receptor alpha chain in a prior study indicated that the mAbs recognize specific peptide epitopes that are present within the extracellular domain of canine IL-4 receptor alpha [US2018/0346580]. Similar analysis performed for the c152H11-H3L3 antibody to canine IL-4 R alpha, displayed in FIG. 4 , resulted in the identification of amino acid sequences SEQ ID NO: 46 and SEQ ID NO: 47 for epitope(s) that have a reasonable similarity to those previously identified. In addition, as displayed in FIG. 4 , amino acid residues S₁₁₁, H₁₁₂, T₁₁₃, T₁₁₉, Y₁₂₂, T₁₂₄, H₁₂₇ Y₁₅₀, T₁₅₃, Y₁₅₄, T₁₅₈, R₁₆₀, S₁₆₄, T₁₆₅, S₁₆₈ S₁₇₁, Y₁₇₂, and S₁₇₃ were identified as particular contact points [see e.g., SEQ ID NO: 5, for amino acid residue numbering].

SEQUENCE LISTING TABLE SEQ ID NO: NA AA  1 Canine IL-4 receptor a chain with signal sequence √  2 Canine IL-4 receptor a chain with signal sequence √  3 Canine IL-4 receptor a chain without signal sequence √  4 Canine IL-4 receptor a chain without signal sequence √  5 Canine IL-4 receptor a extracellular domain √  6 IgG-A hinge region √  7 IgG-B hinge region √  8 IgG-C hinge region √  9 Modified IgG-D hinge region √ 10 Canine IgG-Bm √ 11 146 E2 HCDR1 √ 12 146 E2 HCDR1 √ 13 146 E2 HCDR2 √ 14 146 E2 HCDR2 √ 15 146 E2 HCDR3 √ 16 146 E2 HCDR3 √ 17 146 E2 LCDR1 √ 18 146 E2 LCDR1 √ 19 146 E2 LCDR2 √ 20 146 E2 LCDR2 √ 21 146 E2 LCDR3 √ 22 146 E2 LCDR3 √ 23 152 H11 HCDR1 √ 24 152 H1l HCDR1 √ 25 152 H11 HCDR2 √ 26 152 H11 HCDR2 √ 27 152 H11 HCDR3^(#) √ 28 152 H11 HCDR3^(#) √ 29 152 H11 LCDR1 √ 30 152 H11 LCDR1 √ 31 152 H11 LCDR2 √ 32 152 H11 LCDR2 √ 33 152 H11 LCDR3 √ 34 152 H11 LCDR3 √ 35 C152H11VL3-cCLk-s (kappa light chain) √ 36 C152H11VHl-cIgG-Bm (heavy chain) √ 37 C152H11VH2-cIgG-Bm (heavy chain) √ 38 C152H11VH3-cIgG-Bm (heavy chain) √ 39 cl46E2VL3-cCLk-s (kappa light chain) √ 40 cl46E2VH1-cIgG-Bm (heavy chain) √ 41 cl46E2VH2-cIgG-Bm (heavy chain) √ 42 cl46E2VH3-cIgG-Bm (heavy chain) √ 43 C152H11LV1-cC1 (lambda light chain) √ 44 cl46E2LV1-cC1 (lambda light chain) √ 45 Canine IgG-B √ 46 ISHTWLLMWTNPYPTENHLHS √ 47 NDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWA √ 48 152 H11 HCDR3 √ 49 152 H11 HCDR3 √ ^(#)The C-terminal cysteine residue of HCDR3 of 152H11 was replaced with a serine residue. 

1. An isolated mammalian antibody or antigen binding fragment thereof that binds canine interleukin-4 receptor α (IL-4R_(α)) comprising a set of six complementary determining regions (CDRs), three of which are heavy chain CDRs: a CDR heavy 1 (HCDR1), a CDR heavy 2 (HCDR2), and a CDR heavy 3 (HCDR3) and three of which are light chain CDRs: a CDR light 1 (LCDR1), a CDR light 2 (LCDR2), and a CDR light 3 (LCDR3); wherein (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 26; (iii) the HCDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 28 and SEQ ID NO: 49; (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 30; (v) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32; and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO:
 34. 2. The isolated mammalian antibody or antigen binding fragment thereof of claim 1, wherein the antibody and antigen binding fragment thereof bind canine IL-4R_(α) and block the binding of canine IL-4R_(α) to canine interleukin-4.
 3. The isolated mammalian antibody or antigen binding fragment thereof of claim 1, that is a caninized antibody or an antigen binding fragment thereof.
 4. The caninized antibody or antigen binding fragment thereof of claim 3, that comprises a hinge region that comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 5. The caninized antibody or antigen binding fragment thereof of claim 3, that comprises a heavy chain comprising a modified canine IgG-B (IgG-Bm) comprising the amino acid sequence of SEQ ID NO:
 10. 6. The caninized antibody or antigen binding fragment thereof of claim 5, that comprises a heavy chain that comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:
 38. 7. The caninized antibody of claim 6 or antigen binding fragment thereof, that comprises a light chain that comprises the amino acid sequence SEQ ID NO:
 35. 8. The caninized antibody of claim 6 or antigen binding fragment thereof, that comprises a light chain that comprises the amino acid sequence SEQ ID NO:
 43. 9. An isolated nucleic acid that encodes the heavy chain of the caninized antibody or antigen binding fragment thereof of claim
 3. 10. An isolated nucleic acid that encodes the light chain of the caninized antibody or antigen binding fragment thereof of claim
 3. 11. An expression vector comprising the isolated nucleic acid of claim
 9. 12. A host cell comprising the expression vector of claim
 11. 13. A pharmaceutical composition comprising the caninized antibody of claim 3, and a pharmaceutically acceptable carrier or diluent.
 14. A method of aiding in the blocking of inflammation associated with atopic dermatitis, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim
 13. 15. An expression vector comprising the isolated nucleic acid of claim
 10. 16. A host cell comprising the expression vector of claim
 15. 17. A pharmaceutical composition comprising the caninized antibody of claim 8, and a pharmaceutically acceptable carrier or diluent.
 18. A method of aiding in the blocking of inflammation associated with atopic dermatitis, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim
 17. 19. A pharmaceutical composition comprising the caninized antibody of claim 7, and a pharmaceutically acceptable carrier or diluent.
 20. A method of aiding in the blocking of inflammation associated with atopic dermatitis, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim
 19. 